Fungicidal antibiotic producing Streptomyces sp. NCIMB 40212

ABSTRACT

The Antibiotic Complex AB-023 and its components: Antibiotic AB-023a and  ibiotic AB-023b are disclosed, which are obtained by the controlled aerobic culture of Streptomyces sp. NCIMB 40212 in an aqueous nutrient culture substrate. Antibiotics AB-023 display a biological activity against pathogen fungi which attack agricultural crops and man.

This is a Division of application Ser. No. 08/136,754 filed on Oct. 15,1993, now U.S. Pat. No. 5,347,570, which is a Division of applicationSer. No. 07/747,018, filed on Aug. 19, 1991, now U.S. Pat. No.5,279,829.

The present invention relates to antibiotic substances designated as<<Antibiotic Complex AB-023>> and to its components: Antibiotic AB-023aand Antibiotic AB-023b.

The present invention relates furthermore to the process for preparingsaid Antibiotics AB-023, by means of the fermentation of Streptomycessp. NCIMB 40212, and to their use in the treatment of phytopathologiescaused by the fungal species sensible to said antibiotics.

The <<Antibiotic Complex AB-023>> and its components: Antibiotic AB-023aand Antibiotic AB-023b are different from the other antibiotics knownfrom the prior art.

By the term <<Antibiotic Complex AB-023>>, as used in the instantdisclosure, a mixture is meant which comprises all those components,endowed with one or more biological activities, which are produced bythe fermentation of Streptomyces sp. NCIMB 40212 under such conditionsas specified in detail in the following.

Said biologically active components comprise, but are not limited to,those designated as <<Antibiotic AB-023a>> and <<Antibiotic AB-023b>>,isolated from the mixture.

As those skilled in the art of fermentations are well aware of, thenumber and the relative ratio of the components which constitute theAntibiotic Complex AB-023 may vary, depending on the fermentationconditions (such as, e.g., the culture substrate, the fermentationtemperature, the duration of the same fermentation, the aeration, and soforth), and of the bacterial strain used.

One should furthermore understand that the scope of the instantinvention is not limited to the use of Streptomyces sp. NCIMB 40212, butthat it also encompasses the use of variants and/or mutants obtainedeither naturally or artificially from said microorganism, provided thatthey are capable of producing the Antibiotics AB-023.

Therefore, an object of the instant invention is the Antibiotic ComplexAB-023 which can be obtained by cultivating under controlled, aerobic,conditions, Streptomyces sp. NCIMB 40212, or an equivalent thereof whichis a natural or artificial variant or mutant thereof, in an aqueousnutrient culture substrate, containing assimilable carbon and nitrogensources, inorganic salts and metal in trace amounts, and subsequentlyseparating said Antibiotic Complex AB-023 and its Antibiotic AB-023a andAntibiotic AB-023b components.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. ultraviolet spectrum of AB-023

FIG. 2. infrared spectrum of AB-023

FIG. 3. ¹ H NMR spectrum of AB-023

FIG. 4. ¹³ C NMR spectrum of AB-023

FIG. 5. ultraviolet spectrum of AB-023a

FIG. 6. infrared spectrum of AB-023a

FIG. 7. ¹ H NMR spectrum of AB-023a

FIG. 8. ¹³ C NMR spectrum of AB-023a

FIG. 9. ultraviolet spectrum of AB-023b

FIG. 10. infrared spectrum of AB-023b

FIG. 11. ¹ H NMR spectrum of AB-023b

FIG. 12. ¹³ C NMR spectrum of AB-023b

Physical-Chemical Characteristics of Antibiotic Complex AB-023

The Antibiotic Complex AB-023 is mainly constituted by AntibioticAB-023a and Antibiotic AB-023b, and has the appearance of a yellowpowder, characterized by:

(a) good solubility in dimethyl-sulfoxide, methanol, ethanol,methanol:water (1:1, volume/volume [v/v]) mixture and ethanol:water(1:1, v/v) mixture, insolubility in water, hydrocarbons, acetonitrile,methylene chloride, chloroform;

(b) approximate elemental analysis, as determined on a sample kept undervacuum at 40° C. for two hours, as percent values: carbon: 64.06;hydrogen: 8.78; the product does not contain nitrogen, phosphorus andsulfur;

(c) the ultra-violet (U.V.) absorption spectrum is reported in FIG. 1 ofthe accompanying drawings; it shows absorbance peaks at 304.0 nm, 317.2nm, 332.4 nm, 350.4 nm at a concentration of 0.022 mg/ml inethanol:water (50:50 v/v);

(d) the infrared (I.R.) absorption spectrum in KBr tablet is reported inFIG. 2 of the accompanying drawings, and shows the following absorptionpeaks (cm⁻¹): 3402, 3014, 2970, 2936, 2879, 2041, 1725, 1636, 1457,1379, 1311, 1266, 1175, 1109, 1072, 1008, 905, 847, 823, 745, 706, 583,535, 515, 480;

(e) the N.M.R. spectrum of ¹ H is reported in FIG. 3, and shows signalsrecorded with a Bruker Spectrometer AM at 300 MHz inhexadeutero-dimethylsulfoxide (DMSOd6);

the chemical shifts were indirectly referred to TMS=0.00 ppm (TMS) byusing as the internal reference the central peak ofhexadeutero-dimethylsulfoxide taken at TMS=2.56 ppm.

(ppm): 6.43-6.24 (m, 12H); 6.24-6.07 (m, 4H); 5.96 (m, 2H); 5.62 (m,2H); 5.33 (bs, 1H); 5.19 (bs, 1H); 5.03 (bs, 1H); 4.96 (bs, 2H); 4.93(bs, 1H); 4.86 (m, 4H); 4.70 (m, 2H); 4.42 (bs, 2H); 3.94-3.36 (m, 12H);2.41 (m, 2H); 2.32 (m, 1H); 2.20 (m, 1H); 1.92-1.45 (m, 6H); 1.45-1.09(m, 42H); 1.25 (d, 6H); 1.11 (d, 3H); 1.07 (d, 6H); 0.95 (d, 6H); 0.88(t, 3H).

(f) the N.M.R. spectrum of ¹³ C is reported in FIG. 4 and shows signalsrecorded with a Bruker Spectrometer AM at 300 MHz inhexadeutero-dimethylsulfoxide (DMSOd6);

the chemical shifts were indirectly referred to TMS=0.00 ppm (TMS) byusing as the internal reference the central peak ofhexadeutero-dimethylsulfoxide taken at TMS=39.50 ppm.

The data relevant to the multiplicity of the signals were obtained bymeans of DEPT experimental tests at 45°, 90°, and 135°;

(ppm): 173.7 (s); 173.1 (s); 136.5 (d); 136.5 (d); 136.5 (d); 136.3 (d);133.1 (d); 133.1 (d); 132.9 (d); 132.9 (d); 132.7 (d); 132.5 (d); 132.1(d); 131.9 (d); 131.6 (d); 131.4 (d); 131.2 (d); 131.1 (d); 130.6 (d);130.3 (d); 129.5 (d); 129.2 (d); 74.7 (d); 74.4 (d); 74.1 (d); 73.6 (d);72.0 (d); 71.9 (d); 71.5 (d); 71.4 (d); 71.4 (d); 71.2 (d); 69.7 (d);69.6 (d); 67.7 (d); 67.3 (d); 54.1 (d); 46.1 (d); 45.5 (d); 45.4 (d);43.4 (t); 42.9 (t); 41.7 (t); 41.5 (t); 40.1 (d); 39.5 (d); 38.9 (t);38.9 (t); 38.8 (t); 38.7 (t); 38.2 (t); 38.1 (t); 22.5 (t); 22.4 (t);22.1 (t); 19.0 (q); 18.7 (q); 15.9 (q); 15.6 (q); 13.2 (q); 11.3 (q);10.4 (q); 10.2 (q).

(g) the retention time (R_(t)) of Antibiotic Complex AB-023, identifiedas one single peak, is of approximately 3.65 minutes when analysed byHPLC on a reverse-phase column under the following conditions:

* column: Merck's Lichrosorb RP-18, mm 250×4

* precolumn: Merck C18

* eluent: MeOH:H₂ O (86:14 v/v)

* flowrate: 1 ml/minute

* detector: U.V., 333 nm

* temperature: 35° C.

The retention times of the individual antibiotic components: AB-023a andAB-023b, as resolved peaks, is of 6.18 minutes and 6.76 minutes,respectively, when analysed by HPLC on a reverse-phase column under thefollowing conditions:

* column: Merck's Lichrosorb RP-18, mm 250×4

* precolumn: Merck C18

* eluent: acetonitrile:H₂ O (52.48 v/v)

* flowrate: 1 ml/minute

* detector: U.V., 333 nm

* temperature: 35° C.

Physical-Chemical Characteristics of Antibiotic AB-023a

The Antibiotic AB-023a has the appearance of a yellow powder,characterized by:

(a) good solubility in dimethyl-sulfoxide, methanol, ethanol,methanol:water (1:1, v/v) mixture and ethanol:water (1:1, v/v) mixture,insolubility in water, hydrocarbons, acetonitrile, methylene chloride,chloroform;

(b) approximate elemental analysis, as determined on a sample kept undervacuum at 40° C. for two hours, as percent values: carbon: 63.88;hydrogen: 8.64; the product does not contain nitrogen, phosphorus andsulfur;

(c) the molecular weight of Antibiotic AB-023a is of 550 Da, as it canbe inferred from its FAB-MS spectrum, showing a peak at m/z=551 Da,corresponding to [MH]⁺ ion, and a peak at m/z=549 Da, corresponding to[M--H]⁻ ion. The operating conditions are as follows:

FAB negative ions and positive ions, Xe at 9.5 KV, matrix glycerol,

Finning Mat 8424;

(d) the most probable minimal formula of Antibiotic AB-023a results tobe: C₃₁ H₅₀ O₈ as deduced from the data from ¹ H--N.M.R. and ¹³C--N.M.R. spectroscopy and from mass spectrometry.

(e) the ultra-violet (U.V.) absorption spectrum is reported in FIG. 5 ofthe accompanying drawings; it shows absorbance peaks at 303.0 nm(=22678), 316.6 nm (=44979), 331.6 nm (=71198), 349.8 nm (=71952), at aconcentration of 0.073 mg/ml in ethanol;

(f) the infrared (I.R.) absorption spectrum in KBr tablet is reported inFIG. 6 of the accompanying drawings, and shows the following absorptionpeaks (cm⁻¹):

3402, 3014, 2970, 2936, 2879, 2041, 1725, 1636, 1457, 1379, 1311, 1266,1175, 1109, 1072, 1008, 905, 847, 823, 745, 706, 583, 535, 515, 480;

(e) the N.M.R. spectrum of ¹ H is reported in FIG. 7, and shows signalsrecorded with a Bruker Spectrometer AM at 300 MHz inhexadeutero-dimethylsulfoxide (DMSOd6); the chemical shifts wereindirectly referred to TMS=0.00 ppm (TMS) by using as the internalreference the central peak of hexadeutero-dimethylsulfoxide taken atTMS=2.56 ppm (the more largely overlapped signals were identified bymeans of bidimensional-N.M.R. techniques);

(ppm): 6.32 (m, 6H); 6.17 (m, 1H); 6.16 (m, 1H); 5.93 (dd, 1H); 5.64(dd, 1H); 5.18 (d, 1H); 4.96 (d, 1H); 4.87-4.85 (m, 3H); 4.70 (m, 1H);4.43 (d, 1H); 3.86 (m, 1H); 3.77 (m, 1H); 3.68 (m, 1H); 3.57 (m, 1H);3.51 (m, 1H); 3.48 (m, 1H); 2.40 (m, 1H); 2.32 (m, 1H); 1.68 (m, 1H);1.46-1.09 (m, 14H); 1.24 (d, 3H); 1.11 (d, 3H); 1.07 (d, 3H); 0.93 (d,3H).

(h) the N.M.R. spectrum of ¹³ C is reported in FIG. 8 and shows signalsrecorded with a Bruker Spectrometer AM at 300 MHz inhexadeutero-dimethylsulfoxide (DMSOd6); the chemical shifts wereindirectly referred to TMS=0.00 ppm (TMS) by using as the internalreference the central peak of hexadeutero-dimethylsulfoxide taken atTMS=39.50 ppm.

The data relevant to the multiplicity of the signals were obtained bymeans of DEPT experimental tests at 45°, 90°, and 135°;

(ppm): 173.7 (s); 136.5 (d); 136.3 (d); 132.9 (d); 132.9 (d); 132.5 (d);132.1 (d); 131.6 (d); 131.2 (d); 130.3 (d); 129.5 (d); 74.4 (d); 73.6(d); 72.0 (d); 71.4 (d); 71.2 (d); 69.6 (d); 67.7 (d); 46.1 (d); 45.4(d); 43.4 (t); 41.5 (t); DMSO*; DMSO*; DMSO*; 38.2 (t); 22.4 (t); 18.7(q); 15.9 (q); 13.2 (q); 10.4 (q);

(*): these peaks are covered by the strong signal generated by thesolvent;

(i) the retention coefficients in thin-layer chromatography with aneluent run of 15 cm on reverse-phase silica slabs RP-18 F 254(Merck-Schuchardt) and on reverse-phase silica slabs RP-8 F 254(Merck-Schuchardt), in the following eluent systems are:

* A eluent=water

* B eluent=acetonitrile

* C eluent=methanol

* D eluent=10 mM solution of KH₂ PO₄ in H₂ O adjusted at pH=3 with H₃PO₄ ;

    ______________________________________                                        A:B 50:50 (v/v)    C:D 70:30 (v/v)                                            eluent system      eluent system                                              ______________________________________                                        RP-8     RP-18         RP-8    RP-18                                          0, 30    0, 16         0, 14   0.00                                           ______________________________________                                    

Visualization:

(1) fluorescence under UV-light (355 nm)

(2) reactant prepared by dissolving 20 g of (NH₄)₃ MoO₄ and 1 g ofCe(SO₄)₂ in an aqueous solution containing 6% of H₂ SO₄ ;

(1) the retention time of Antibiotic AB-023a is of approximately 6.18minutes when analysed by HPLC on a reverse-phase column under thefollowing conditions:

* column: Merck's Lichrosorb RP-18, mm 250×4

* precolumn: Merck C18

* eluent: Acetonitrile:H₂ O (52:48 v/v)

* flowrate: 1 ml/minute

* detector: U.V., 333 nm

* temperature: 35° C.

Physical-Chemical Characteristics of Antibiotic AB-023b

The Antibiotic AB-023b has the appearance of a yellow powder,characterized by:

(a) good solubility in dimethyl-sulfoxide, methanol, ethanol,methanol:water (1:1, v/v) mixture and ethanol:water (1:1, v/v) mixture,insolubility in water, hydrocarbons, acetonitrile, methylene chloride,chloroform;

(b) approximate elemental analysis, as determined on one sample keptunder vacuum at 40° C. for two hours, as percent values: carbon: 65.18;hydrogen: 8.87; the product does not contain nitrogen, phosphorus andsulfur;

(c) the molecular weight of Antibiotic AB-023b is of 564 Da, as it canbe inferred from its FAB-MS spectrum, showing a peak at m/z=565 Da,corresponding to [MH]⁺ ion, and a peak at m/z=563 Da, corresponding to[M--H]⁻ ion. The operating conditions are as follows:

FAB negative ions and positive ions, Xe at 9.5 kV, matrix glycerol,

Finning Mat 8424;

(d) the most probable minimal formula of Antibiotic AB-023b results tobe: C₃₂ H₅₂ O₈ as deduced from the data from ¹ H-N.M.R. and ¹³ C-N.M.R.spectroscopy and from mass spectrometry.

(e) the ultra-violet (U.V.) absorption spectrum is reported in FIG. 5 ofthe accompanying drawings; it shows absorbance peaks at 303.3 nm(=27536), 317.0 nm (= 72192), 332.0 nm (=90562), 348.5 nm (=86534), at aconcentration of 0.0070 mg/ml in ethanol;

(f) the infrared (I.R.) absorption spectrum in KBr tablet is reported inFIG. 10 of the accompanying drawings, and shows the following absorptionpeaks (cm⁻¹):

3402, 3014, 2970, 2936, 2879, 2041, 1725, 1636, 1457, 1379, 1311, 1266,1175, 1109, 1072, 1008, 905, 847, 823, 745, 706, 583, 535, 515, 480;

(e) the N.M.R. spectrum of ¹ H is reported in FIG. 11, and shows signalsrecorded with a Bruker Spectrometer AM at 300 MHz inhexadeutero-dimethylsulfoxide (DMSOd6);

the chemical shifts were indirectly referred to TMS=0.00 ppm (TMS) byusing as the internal reference the central peak ofhexadeutero-dimethylsulfoxide taken at TMS=2.56 ppm (the signals morelargely overlapped were identified by means of bidimensional-N.M.R.techniques);

(ppm): 6.43-6.22 (m, 6H); 6.22-6.10 (m, 2H); 5.97 (dd, 1H); 5.60 (dd,1H); 5.33 (bs, 1H); 5.03 (bs, 1H); 4.96 (bs, 1H); 4.92 (bs, 1H); 4.85(m, 1H); 4.70 (m, 1H); 4.42 (bs, 1H); 3.94-3.36 (m, 6H); 2.41 (m, 1H);2.32 (m, 1H); 2.20 (m, 1H); 1.92-1.45 (m, 6H); 1.45-1.09 (m, 8H); 1.25(d, 3H); 1.07 (d, 3H); 0.95 (d, 3H); 0.88 (t, 3H);

(h) the N.M.R. spectrum of ¹³ C is reported in FIG. 12 and shows signalsrecorded with a Bruker Spectrometer AM at 300 MHz inhexadeutero-dimethylsulfoxide (DMSOd6);

the chemical shifts were indirectly referred to TMS=0.00 ppm (TMS) byusing as the internal reference the central peak ofhexadeutero-dimethylsulfoxide taken at TMS=39.50 ppm.

The data relevant to the multiplicity of the signals were obtained bymeans of DEPT experimental tests at 45°, 90°, and 135°;

(ppm): 173.3 (d); 136.7 (d); 136.7 (d); 133.2 (d); 133.2 (d); 132.7 (d);132.0 (d); 131.6 (d); 131.3 (d); 130.7 (d); 129.4 (d); 74.8 (d); 74.3(d); 72.1 (d); 71.6 (d); 71.5 (d); 69.9 (d); 67.5 (d); 54.3 (d); 45.6(d); 43.1 (t); 41.9 (t); DMSO; DMSO; DMSO; DMSO; 22.7 (t); 22.2 (t);19.1 (q); 15.6 (q); 11.5 (q); 10.4 (q);

(i) the retention coefficients in thin-layer chromatography with aneluent run of 15 cm on reverse-phase silica slabs RP-18 F 254(Merck-Schuchardt) and on reverse-phase silica slabs RP-8 F 254(Merck-Schuchardt), in the following eluent systems are:

* A eluent=water

* B eluent=acetonitrile

* C eluent=methanol

* D eluent=10 mH solution of KH₂ PO₄ in H₂ O adjusted at pH=3 with H₃PO₄ ;

    ______________________________________                                        A:B 50:50 (v/v)    C:D 70:30 (v/v)                                            eluent system      eluent system                                              ______________________________________                                        RP-8     RP-18         RP-8    RP-18                                          0, 28    0, 16         0, 12   0.00                                           ______________________________________                                    

Visualization:

(1) fluorescence under UV-light (366 nm)

(2) reactant prepared by dissolving 20 g of (NH₄)₃ MoO₄ and 1 g ofCe(SO₄)₂ in an aqueous solution containing 6% of H₂ SO₄ ;

(1) the retention time of Antibiotic AB-023b is of approximately 6.76minutes when analysed by HPLC on a reverse-phase column under thefollowing conditions:

* column: Merck's Lichrosorb RP-18, mm 250×4

* precolumn: Merck C18

* eluent: Acetonitrile:H₂ O (52:48 v/v)

* flowrate: 1 ml/minute

* detector: U.V., 333 nm

* temperature: 35° C.

Morphology and cultural characteristics of the microorganismStreptomyces sp. NCIMB 40212.

The microorganism Streptomyces sp. NCIMB 40212 was isolated from a soilsample collected at Nairobi (Kenia) and recorded, for internallaboratory use, with the conventional code SD-581.

On Oct. 3rd, 1989, a culture of this microorganism was deposited, inpursuance of the Budapest Covenant, with the National Collection ofIndustrial Bacteria (c/o The National Collection of Industrial andMarine Bacteria, Ltd., Torrey Research Station, P.O. Box 31, Abbey Road,Aberdeen AB DG-U.K.), were it was assigned the NCIMB access number40212.

The morphological characteristics of the microorganism are reported infollowing Table A (the culture substrates are indicated with their nameas reported in International Streptomyces Program).

In Tables B and C, some characteristics concerning the culture of thisstrain are reported.

                                      TABLE A                                     __________________________________________________________________________    Morphology on several culture substrates                                                 ISP                                                                              Growth                                                          Substrate  (1)                                                                              (2)  Base Mycelium                                                                            Aerial Mycelium                                                                           Soluble Pigment                     __________________________________________________________________________    Malt extract                                                                             M2 +++  raised, light colour                                                                     abundant, gray colour                                                                     -                                   Dat Meal Agar                                                                            M3 ++   smooth, yellow colour                                                                    scarce, gray colour                                                                       -                                   Starch Agar                                                                              M4 +++  raised, light colour                                                                     abundant, gray colour                                                                     -                                   Glycerol Asparagine                                                                      M5 +++  smooth, yellow colour                                                                    abundant, gray colour                                                                     -                                   Peptone Yeast Extract                                                                    M6 ++   smooth, yellow colour                                                                    abundant, gray colour                                                                     -                                   Tyrosine Agar                                                                            M7 ++   smooth, light colour                                                                     scarce, white colour                                                                      -                                   Czapek Peptone                                                                              ++   smooth, light colour                                                                     scarce, gray colour                                                                       -                                   Dextrose Potato                                                                             +++  smooth, light colour                                                                     abundant, gray colour                                                                     -                                   Nutrient Agar +++  smooth, yellow colour                                                                    abundant, white colour                                                                    light, yellow colour                Water Agar    ++   smooth, light colour                                                                     scarce, gray colour                                                                       -                                   __________________________________________________________________________

                  TABLE B                                                         ______________________________________                                        Antibiogram (4)                                                               Antibiotic       Dose (g) Sensibility                                         ______________________________________                                        Nalidixic Acid   30       ++                                                  Ampicillin       10       -                                                   Bacitracin       10       ++                                                  Cefaloridin      30       -                                                   Chloramphenicol  30       +                                                   Chlorotetracycline                                                                             30       +                                                   Erythromycin     15       +++                                                 Fosfomycin       50       -                                                   Gentamycin       10       +                                                   Kanamycin        30       ++                                                  Lincomycin        2       -                                                   Neomycin         30       +                                                   Novobiocin       30       +++                                                 Oleandomycin     15       +++                                                 Oxytetracycline  30       +                                                   Penicillin       10       -                                                   Rifamycin        30       ++                                                  Rifampicin       30       +++                                                 Streptomycin     10       +                                                   Tetracycline     30       +                                                   Tobramycin       10       +                                                   Vancomycin       30       ++                                                  ______________________________________                                    

                  TABLE C                                                         ______________________________________                                        Auxanogram (3)                                                                Compound           Growth (2)                                                 ______________________________________                                        Adonitol           -                                                          L-Arabinose        -                                                          Cellobiose         -                                                          Galactose          -                                                          Glucose            +                                                          Glycerol           +++                                                        Inositole          -                                                          Lactose            -                                                          Maltose            +++                                                        Methyl-D-glucoside -                                                          Melezitose         -                                                          N-Acetyl-D-Glucosamine                                                                           +++                                                        Raffinose          -                                                          Saccharose         -                                                          Sorbitol           -                                                          Trehalose          -                                                          Xylitol            -                                                          Xylose             -                                                          2-Keto-D-Gluconate -                                                          ______________________________________                                         Remarks:                                                                      (1) Code assigned by the International Streptomyces Program.                  (2) + = poor;                                                                 ++ = good;                                                                    +++ = abundant.                                                               (3) Performed by using the API systems SA's API:API 200 Kit.                  (4) Performed by using the SensiDisc (of 5 mm of diameter), marketed by       BBL (Becton Dickinson Microbiology Systems).                             

The analysis by scanning electron microscopy shows that the mycelium ofStreptomyces sp. NCIMB 40212 tends to form filaments constituted by aplurality of twisted Hyphae and that the spore chains are ofrectiflexible type, with smooth spores. These features assign NCIMB40121 strain to Streptomyces genus.

Like other microorganisms, Streptomyces sp. NCIMB 40212 may undergoeither natural or artificial variations and/or mutations.

For example, artificial variants or mutants can be obtained by treatingthe microorganism with chemical mutagenic agents such as, e.g., nitrousacid, nitroso-guanidine, halogenated alkyl-amines and the like, or withsuch physical agents as X rays, ultraviolet light (U.V.), orhigh-frequency waves.

Natural variants or mutants can be obtained as well, by isolating andselecting different colonies obtained from Streptomyces sp. NCIMB 40212.

All either natural or artificial variants and/or mutants belonging toStreptomyces genus and producing AB-023 Antibiotics are considered asequivalent to microorganism Streptomyces sp. NCIMB 40212 and fall withinthe scope of the instant invention.

Process for preparing Antibiotics AB-023

The process for preparing the Antibiotics AB-023 consists in cultivatingStreptomyces sp. NCIMB 40212, or a natural or artificial variantthereof, under controlled aerobic fermentation conditions, in an aqueousnutrient substrate and separating said AB-023 Antibiotics by means ofper se known methods.

As the nutrient culture substrates, those substrates can be used, whichare customarily used for producing antibiotics; however, some nutrientculture media will be preferred.

Said culture substrates must contain carbon and nitrogen sources whichmust be assimilable by the microorganisms belonging to Streptomycesgenus; they must furthermore contain low concentrations of inorganicsalts, and traces of those metals which are necessary for the growth anddevelopment of the microorganisms, and for the production ofantibiotics. Said metal traces may be already present, as impurities, inthe organic nitrogen and carbon sources included in the culturesubstrates in order to allow the bacterial growth, or, whenevernecessary, can be added to said culture substrates.

In general, as the source of assimilable carbon, there can be usedcarbohydrates, in particular, saccharides, such as, e.g., dextrose,maltose, lactose, or, either alternatively or as a complement, starchesand those industrial products which are chemically related to starches,such as, e.g., dextrose or soluble starch, or also polyhydroxy alcohols,such as, e.g., glycerol. Said compounds can be used either as individualcompounds, or as combinations thereof, in variable mutual proportions.

The concentration and the type of carbon source contained in the culturesubstrate depend in general on the type and amount of the othercomponents of the substrate; however, concentrations comprised withinthe range of from 0.5 to 5% by weight are satisfactory.

As the source of organic nitrogen, there can be used proteinic extractssuch as, e.g., yeast extract, meat extract and casein hydrolysate, ormeals, such as, e.g., soy bean meal, or industrial products marketed forthis purpose, such as, e.g. <<Proflo>>, <<Corn Steep Liquor>> and<<Distillers Solubles>>. These compounds can be used eitherindividually, or as combinations thereof, in variable proportions. Theirconcentrations in the culture substrate may be comprised within therange of from 0.2 to 6% by weight.

As regards the inorganic salts, there can be used, e.g., such sodium,potassium, magnesium, iron, ammonium and calcium salts, as phosphates,sulfates, chlorides, carbonates and nitrates.

The metals present in trace amounts in the culture substrate can be,e.g., cobalt, iron, manganese, and the like.

Some culture substrates have displayed a capability of stimulating theproduction of Antibiotics AB-023 by Streptomyces sp. NCIMB 40212. Amongthem, the aqueous formulations, which are used in the following examplesof preparation, can be mentioned:

    ______________________________________                                        (Ingredients)        (concentration, g/l)                                     ______________________________________                                        <<A>> Culture Substrate (PM8)                                                 *Starch              20                                                       *Glucose             10                                                       *Calcium carbonate   3                                                        *Hydrolysed casein   2                                                        *Proflo              2                                                        *Yeast extract       2                                                        *Meat extract        2                                                        pH                   7,0                                                      <<B>> Culture Substrate (PGC)                                                 *Glycerol            30                                                       *Proflo              20                                                       *CaCO.sub.3          3                                                        pH                   6.5-7.0                                                  ______________________________________                                    

The microorganism Streptomyces sp. NCIMB 40212 can be cultivated andfermented in order to produce Antibiotics AB-023, at temperaturescomprised within the range of from 20° C. to 35° C., preferably of from25° C. 30° C.

The pH values can generally be comprised within the range of from about5 to about 9.

The sterile air blown through the culture substrate is used in generalin such amounts, as to maintain in the substrate an oxygen concentrationof more than 20% of saturation concentration.

The production of Antibiotics AB-023, during the fermentation, can bemonitored by checking the biological activity on broth samples, usingthe fungal species sensible to the same Antibiotics.

The fermentation is continued for a long enough time, until asubstantial antibiotic activity is achieved: time periods comprisedwithin the range of from 24 to 150 hours are generally enough.

Separation and purification of Antibiotic Complex AB-023 and itsAntibiotic AB-023a and Antibiotic AB-023b components

After the cultivation under the above disclosed fermentation conditions,the Antibiotic Complex AB-023 can be separated from the culture broth,and subsequently purified, by means of conventional methods known fromfermentation art.

Such methods comprise, e.g., the extraction with solvents, precipitationwith non-solvents, ultrafiltration, column chromatography in direct- orreverse-phase mode, chromatography on non-ionic or ionic macroporousresins, and the like.

The antibiotics produced during the fermentation are contained in themyceliar mass.

A preferred method in order to recover the Antibiotic Complex AB-0232consists in centrifuging off the myceliar mass from the culture broth,submitting the so separated mycelium to an extraction with ethanol oraqueous ethanol, separating the exhausted mycelium from the alcoholicextract by paper filtration, and vacuum-concentrating the extract todryness, in order to obtain a raw product.

The Antibiotic Complex AB-023 is then isolated from said raw product, byadsorption on non-ionic polystyrene resin, for example, of XAD-4 type(available from Rohm and Haas Co.). The resin is then washed with 2volumes--as referred to the volume of the resin bed--of water, and thenis eluted with 3 volumes--also as referred to the volume of the resinbed--of an eluent system formed by an <<A>> eluent consisting of water,and a <<B>> eluent constituted by methanol:acetonitrile (50:50, v/v),with a gradient of from 20% to 80% of <<B>> eluent.

The fractions which contain the Antibiotic Complex AB-023, identified bybiological tests for activity on Botrytis, are chromatographed by usinga column packed with reverse-phase silica of MATREX Silica C18 type(available from Amicon Europe, Lausanne, Switzerland), with an eluentsystem formed by an <<A>> eluent consisting of water, and a <<B>> eluentconstituted by acetonitrile, with a linear gradient of from 20% to 80%of the <<B>> eluent in the <<A>> eluent.

The fractions which contain the Antibiotic Complex AB-023 are combinedand concentrated under vacuum, until no acetonitrile is present anylonger. From the so-obtained aqueous suspension, the Antibiotic ComplexAB-023 precipitates by cooling, as a solid, yellow-coloured substance.

After centrifuging off the aqueous supernatant, and drying theprecipitate under the vacuum of the rotary pump, the Antibiotic Complexdesignated AB-023 is obtained, which is prevailingly constituted byAntibiotics AB-023a and AB-023b, in a mutual ratio of about 1:1.

The individual AB-023a and AB-023b antibiotics can be separated fromeach other by submitting the Antibiotic Complex AB-023 to a furtherchromatographic separation, on a reverse-phase silica column of MATREXSilica C18 type (available from Amicon Europe, Lausanne, Switzerland),with an eluent system formed by an <<A>> eluent consisting of water, anda <<B>> eluent constituted by acetonitrile, with a gradient of from 20%to 80% of the <<B>> eluent in the <<A>> eluent.

Biological Activity

The Antibiotic Complex AB-023 and its Antibiotic AB-023a and AntibioticAB-023b components display an antifungal activity, against the pathogenfungi which infest the cereal crops, fruit trees and horticulturalcrops, as well as against pathogen fungi which attack man.

The antifungal activities in vitro of Antibiotics AB-023 were determinedby the methods disclosed hereinafter.

Test for <<in vitro>> fungicidal activity

The antimicrobial activity of the Antibiotics AB-023 is determined bymeans of the usual methods, by adding increasing concentrations of theantibiotic to an agarised substrate capable of supporting the growth ofthe sensible microbial species. For phytopathogen fungi, the minimumconcentration--expressed as ppm--was determined of Antibiotics AB-023which, under these conditions, completely inhibits the growth (MinimalInhibitory Concentration, MIB). In Table D, the data of biologicalactivity are reported, which were obtained in vivo by means of themethods described hereinabove.

                  TABLE D                                                         ______________________________________                                                         Minimal Inhibitory                                           TEST FUNGUS      Concentration, ppm                                           ______________________________________                                        Botrytis cinerea 5                                                            Helminthosporium teres                                                                         10                                                           Fusarium roseum  10                                                           Fusarium moniliforme                                                                           25                                                           Rhizoctonia solani                                                                             10                                                           Colletotricum coffeanum                                                                        10                                                           Piricularia orizae                                                                             10                                                           Pithium ultimum  100                                                          Candida albicans 10                                                           Sarcina lutea    100                                                          ______________________________________                                    

Very similar results of antifungal activity are obtained by using theindividual components Antibiotic AB-023a and Antibiotic AB-023b.

For the purposes of their practical use, both in agriculture and inother use sectors, the antibiotics according to the present inventionare advantageously used as suitable compositions.

These compositions contain, besides the antibiotic of the invention astheir active principle, inert carriers, which may be either solidsubstances (such as, e.g., China clay, silica, talc, actapulgite,diatomaceous earth, and so forth) or liquid substances (e.g., organicsolvents, vegetable or mineal oils, water, and mixtures thereof), and,possibly, such other additives as normally used in the formulationfield, such as surface active agents, suspending agents, dispersants andwetting agents.

For particular formulation requirements, or in order to expand theactivity range of the compositions, to said compositions, as disclosedhereinabove, further active ingredients may be added, such as, e.g.,insecticides, herbicides and fungicides.

The application doses may vary depending on several factors, such as thetype and level of infestation, the type of composition used, weather andenvironmental factors.

For practical uses in agriculture, doses of AB-023 antibiotics comprisedwithin the range of from 10 to 500 g/hectare yield satisfactory results.

Some examples are given now for the purpose of illustrating theinvention without limiting it.

EXAMPLE 1

A culture of Streptomyces sp. NCIMB 40212 grown on a slant agar-<<A>>substrate (PM8) culture, was re-suspended in 5 ml of sterile distilledwater. Such a suspension was inoculated into an Erlenmeyer flaskcontaining 100 ml of <<A>> substrate (PM8). The Erlenmeyer flask wasthen kept shaked at 180 rpm, at 28° C., for 55 hours. The resultingculture was used to inoculate a matras containing 1 liter of <<A>>substrate (PM8), which was subsequently kept shaked at 150 rpm, at 28°C. for 40 hours. With the resulting culture, a fermentor with a ratedvolume of 40 liters, containing 26 liters of <<B>> substrate (PGC), wasthen inoculated. The fermentation was sustained for 24 hours, with thetemperature being kept at 28° C. and the concentration of dissolvedoxygen being kept higher than 20% of saturation concentration.

EXAMPLE 2

A culture of Streptomyces sp. NCIMB 40212 grown on a slant agar-<<A>>substrate (PM8) culture, was re-suspended in 6 ml of sterile distilledwater. Such a suspension was inoculated into three Erlenmeyer flasks,each of which contained 100 ml of <<A>> substrate (PM8). The Erlenmeyerflasks were then kept shaked at 180 rpm, at 28° C., for 48 hours. Theresulting culture was used to inoculate 50 Erlenmeyer flasks, each ofwhich contained 100 ml of <<B>> substrate (PGC), with an inoculum of 5%by volume. The Erlenmeyer flasks were kept stirred at 180 rpm, at 28° C.for 72 hours. The so obtained broth was then collected and separatedfrom the mycelium by centrifugation at 6,000 rpm for 20 minutes.

Isolation of Antibiotic Complex AB-023

10 liters of fermentation broth were separated from the mycelium, bycentrifugation. The separated mycelium was extracted with aqueousethanol, then the ethanolic exctract of mycelium was vacuum-concentrateddown to a small volume, and was percolated on a non-ionic polystyrenecolumn, of XAD-4 type (available from Rohm and Haas Co.). The resin waswashed with 2 volumes, as referred to the same resin bed volume, ofwater, and was then eluted with 3 volumes, also referred to the sameresin bed volume, of an eluent system formed by an <<A>> eluentconsisting of water, and a <<B>> eluent consisting ofmethanol:acetonitrile (50:50, v/v), with a gradient of from 20% to 80%of the <<B>> eluent in the <<A>> eluent. The fractions containing theAntibiotic Complex AB-023 were combined and concentrated to dryness, inorder to yield a raw product.

Said raw product was dissolved again with 50 ml of a water:acetonitrilemixture (20:80, v/v), with which a reverse-phase silica column wasconditioned (MATREX Silica C-18, Amicon Europe, Lausanne, Switzerland)(diameter of the column 33 mm, height of silica bed 30 cm). TheAntibiotic Complex AB-023 was then eluted from the above saidchromatographic column by using an eluent system formed by acetonitrileand water, with a gradient comprised within the range of from 20% to 80%of acetonitrile, with a volume of 300 ml of eluent being used each time.The fractions containing the Antibiotic Complex AB-023, identified bymeans of the biological test on Botrytis, were combined andvacuum-concentrated, until no acetonitrile was detected any longer.

From the resulting aqueous suspension the Antibiotic Complex designated<<AB-023>> precipitated on cooling, as a yellow-coloured solid. Aftercentrifugation and drying, 300 mg of Antibiotic Complex AB-023,prevailingly formed by Antibiotic AB-023a and Antibiotic AB-023b, in anapproximate mutual ratio of 1:1, was obtained.

Isolation of Antibiotic AB-023a and Antibiotic AB-023b

Antibiotic AB-023a and Antibiotic AB-023b were isolated from AB-023complex by repeating a chromatography on reverse-phase silica column(MATREX Silica C-18, Amicon Europe, Lausanne, Switzerland) (diameter ofthe column 22 mm, height of silica bed 50 cm), by using an eluent systemformed by an <<A>> eluent consisting of water, and a <<B>> eluentconsisting of acetonitrile, with a linear gradient comprised within therange of from 20% to 80% of the <<B>> eluent in the <<A>> eluent.

Some fractions which contained pure Antibiotic AB-023a, and somefractions which contained pure Antibiotic AB-023b were obtained, besidessome fractions which contained the mixture of both, as AB-023 Complex.By concentrating to dryness, under vacuum, the fractions which containedAntibiotic AB-023a and Antibiotic AB-023b in pure form, 50 mg ofAntibiotic AB-023a and 15 mg of Antibiotic AB-023b were obtained asyellow-coloured solid substances.

The physical-chemical of the product are reported above in the instantdisclosure.

We claim:
 1. A biologically pure culture of a microorganism Streptomycessp. NCIMB 40212 or a mutant thereof capable of producing the AntibioticComplex AB-023 in isolatable amounts by controlled aerobic fermentationin an aqueous nutrient substrate comprising assimilable carbon andnitrogen sources and inorganic salts, said Antibiotic Complex AB-023being a solid characterized by:(a) good solubility indimethyl-sulfoxide, methanol, ethanol, methanol:water (1:1,volume/volume [v/v]) mixture and ethanol:water (1:1, v/v) mixture,insolubility in water, hydrocarbons, acetonitrile, methylene chloride,chloroform; (b) approximate elemental analysis, expressed as percentvalues: carbon: 64.06; hydrogen: 8.78; the product does not containnitrogen, phosphorus and sulfur; (c) absorbance peaks within theultra-violet range at 304.0 nm, 317.2 nm, 332.4 nm, 350.4 nm at aconcentration of 0.022 mg/ml in ethanol:water (50:50 v/v); (d)absorption peaks within the infrared range (cm⁻¹): 3402, 3014, 2970,2936, 2879, 2041, 1725, 1636, 1457, 1379, 1311, 1266, 1175, 1109, 1072,1008, 905, 847, 823, 745, 706, 583, 535, 515, 480; (e) N.M.R. spectrumof ¹ H showing the following main peaks:(ppm): 6.43-6.24 (m, 12H);6.24-6.07 (m, 4H); 5.96 (m, 2H); 5.62 (m, 2H); 5.33 (bs, 1H); 5.19 (bs,1H); 5.03 (bs, 1H); 4.96 (bs, 2H); 4.93 (bs, 1H) 4.86 (m, 4H); 4.70 (m,2H); 4.42 (bs, 2H); 3.94-3.36 (m, 12H); 2.41 (m, 2H); 2.32 (m, 1H); 2.20(m, 1H); 1.92-1.45 (m, 6H), 1.45-1.09 (m, 42H); 1.25 (d, 6H); 1.11 (d,3H); 1.07 (d, 6H); 0.95 (d, 6H), 0.88 (t, 3H); (f) N.M.R. spectrum of 1°C. showing the following main peaks:(ppm): 173.7 (s); 173.1 (s); 136.5(d); 136.5 (d); 136.5 (d); 136.3 (d); 133.1 (d); 133.1 (d); 132.9 (d);132.9 (d); 132.7 (d); 132.5 (d); 132.1 (d); 131.9 (d); 131.6 (d); 131.4(d); 131.2 (d); 131.1 (d); 130.6 (d); 130.3 (d); 129.5 (d); 129.2 (d);74.7 (d); 74.4 (d); 74.1 (d); 73.6 (d); 72.0 (d); 71.9 (d); 71.5 (d);71.4 (d); 71.4 (d); 71.2 (d); 69.7 (d); 69.6 (d); 67.7 (d); 67.3 (d);54.1 (d); 46.1 (d); 45.5 (d); 45.4 (d); 43.4 (t); 42.9 (t); 41.7 (t);41.5 (t); 40.1 (d); 39.5 (d); 38.9 (t); 38.9 (t); 38.8 (t); 38.7 (t);38.2 (t); 38.1 (t); 22.5 (t); 22.4 (t); 22.1 (t); 19.0 (q); 18.7 (q);15.9 (q); 15.6 (q); 13.2 (q); 11.3 (q); 10.4 (q); 10.2 (q); (g) aretention time (R_(t)) of 3.65 minutes by reverse-phase HPLC on aMerck's Lichrosorb RP-18 column of mm 250×4, precolumn Merck C18, eluentMeOH:H₂ O (86:14 v/v), with a flowrate of 1 ml/minute and at thetemperature of 35° C.
 2. A composition comprising the biologically pureculture of claim 1.